Prostate cancer treatment via synergistic inhibition of aryl hydrocarbon receptor (ahr) and src

ABSTRACT

Simultaneous inhibition of the aryl hydrocarbon receptor (AhR) and SRC abolishing androgen receptor (AR) signaling in prostate cancer cells is disclosed herein as an effective treatment for prostate cancer. Provided herein is a therapeutic composition that comprises an aryl hydrocarbon receptor (AhR) antagonist and an SRC inhibitor. In one embodiment, the therapeutic composition further comprises a pharmaceutical excipient. In one embodiment, the AhR antagonist is CH223191 and the SRC inhibitor is PP2 and the therapeutic composition further comprises a pharmaceutical excipient. Methods of using the therapeutic composition to treat prostate cancer or to inhibit prostate cancer cells are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional patent applicationSer. No. 62/429,654, filed on Dec. 2, 2016, and entitled SimultaneousInhibition of Aryl Hydrocarbon Receptor (AhR) and SRC abolishes androgenreceptor signaling, which is incorporated herein by reference.

GOVERNMENT RIGHTS

Development of the inventions described herein was at least partiallyfunded with government support through NIH grant 2G12MD007590-29 and theU.S. government has certain rights in the invention.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is part of thedescription and is provided in the form of an Annex C/ST.25 text file inlieu of a paper copy, and hereby incoporated by reference into thespecification. The name of the text file containing the Sequence Listingis 0309-016_SequenceListing_PatentIn_ST25.txt. The text file is 3 kb,was created on Dec. 4, 2017, and is being submitted electronically viaEFS-Web.

FIELD OF THE INVENTION

The present disclosure is directed to simultaneous inhibition of ArylHydrocarbon Receptor (AhR) and SRC to abolish androgen receptorsignaling, and methods of using AhR and SRC inhibitors to treat prostatecancer.

BACKGROUND

Prostate cancer is the second leading cause of cancer death among men inWestern countries. Prostate cancer is the most commonly diagnosed cancerin men. An estimated one in six men will be diagnosed with prostatecancer and 1 in 36 will die from the disease. Such rates establishprostate cancer as one of the leading causes of all cancer-relateddeaths in men. Prostate cancer may be treated through a combination ofsurgery, radiation therapy, hormone therapy or chemotherapy. Theoutcomes of the treatments depend on a person's age and other healthproblems, as well as the extent and aggression of the cancer. Treatmentof aggressive prostate cancers may involve surgery, such as radicalprostatectomy, radiation therapy including brachytherapy, such asprostate brachytherapy, external beam radiation therapy, high-intensityfocused ultrasound (HIFU), chemotherapy, oral chemotherapeutic drugs(temozolomide/TMZ), cryosurgery, hormonal therapy, or combinationthereof. Most hormone dependent cancers become resistant to treatmentafter one to three years and resume growth despite hormone therapy.Regarding “hormone-refractory prostate cancer” or “androgen-independentprostate cancer,” the term castration-resistant has replaced “hormonerefractory” because, while they are no longer responsive to castrationtreatment, i.e., the reduction of available androgen/testosterone/DHT bychemical or surgical means, these cancers still show reliance uponhormones for androgen receptor activation.

The cancer chemotherapic, docetaxel, has been used as treatment forcastration-resistant prostate cancer (CRPC) with a median survivalbenefit of 2 to 3 months. A second-line chemotherapy treatment iscabazitaxel. A combination of bevacizumab, docetaxel, thalidomide, andprednisone has also been shown to be effective in the treatment of CRPC.Immunotherapy treatment with sipuleucel-T in CRPC increases survival by4 months. The second line hormonal therapy abiraterone increasessurvival by 4.6 months when compared to placebo. Enzalutamide is anothersecond line hormonal agent with a 5-month survival advantage overplacebo. Both abiraterone and enzalutamide are currently being tested inclinical trials in individuals with CRPC who have not previouslyreceived chemotherapy. Only a subset of people respond to androgensignaling blocking drugs, but certain cells with characteristicsresembling stem cells remain unaffected. Therefore, the desire toimprove outcomes of people with CRPC has resulted in strategies ofincreasing doses further or combination therapy with synergisticandrogen signaling blocking agents. However, these combinations will notaffect stem-like cells that do not exhibit androgen signaling. Thereremains a need to find better treatment for prostate cancer, especiallyCRPC.

SUMMARY

Provided herein is a method of treating prostate cancer in a livingsubject. The method comprises administering an effective amount of oneor more therapeutic compositions into the living subject to reduceandrogen receptor signaling in the living subject to treat prostatecancer. The one or more therapeutic compositions comprise an arylhydrocarbon receptor (AhR) antagonist and a SRC inhibitor. In oneembodiment, the living subject is a human. In one embodiment, the AhRantagonist is 2-Methyl-2H-pyrazole-3-carboxylic acid(2-methyl-4-o-tolylazo-phenyl)-amide (CH223191). In one embodiment, theSRC inhibitor is4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2).In one embodiment, the prostate cancer is castration resistant. In oneembodiment, the living subject has prostate cancer cells and the methoddisclosed herein reduces the proliferation of the prostate cancer cellsin the living subject by at least 80%. In one embodiment, the livingsubject is a human having castration resistant prostate cancer (CRPC)cells, the AhR antagonist is CH223191, the SRC inhibitor is PP2, and themethod reduces the amount of CRPC cells in the living subject by atleast 90%. In one embodiment, the living subject has prostate cancercells and the method includes administering one or more therapeuticcompositions into the living subject to inhibit the proliferation of thecancer cells.

Further provided is a method of inhibiting prostate cancer cells, themethod including contacting the prostate cancer cells with an effectiveamount of one or more therapeutic compositions to reduce androgenreceptor signaling in the prostate cancer cells, wherein the one or moretherapeutic compositions include an aryl hydrocarbon receptor (AhR)antagonist and a SRC inhibitor. In one embodiment, the AhR antagonist isCH223191. In one embodiment, the SRC inhibitor is PP2. In oneembodiment, the prostate cancer cell is castration resistant. In anotherembodiment, the proliferation of the prostate cancer cells is reduced byat least 80%. In yet another embodiment, the cancer cells are CRPCcells, the AhR antagonist is CH223191, the SRC inhibitor is PP2, and themethod reduces the proliferation of the CRPC cells by at least 90%.

Additionally provided herein is a therapeutic composition having an arylhydrocarbon receptor (AhR) antagonist and a SRC inhibitor. In oneembodiment, the AhR antagonist is CH223191. In one embodiment, the SRCinhibitor is PP2. In one embodiment, the AhR antagonist is CH223191 andthe SRC inhibitor is PP2. In one embodiment, the therapeutic compositionincludes a pharmaceutical excipient. In one embodiment, the AhRantagonist is CH223191 and the SRC inhibitor is PP2 and the therapeuticcomposition further having a pharmaceutical excipient.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawing figures are not necessarily to scale and certain featuresmay be shown exaggerated in scale or in a somewhat generalized orschematic form in the interest of clarity and conciseness. For morecomplete understanding of the features and advantages of the presentinvention, reference is now made to the detailed description of theinvention along with the accompanying figures, wherein:

FIG. 1A shows SDS polyacrylamine gel electrophoresis results of totalcellular proteins of C4-2 prostate cancer cells treated with CH223191and PP2, alone and in combination, with DMSO as a vehicle control.

FIG. 1B shows nuclear and cytoplasmic fractionation of C4-2 cells grownon 100 mm dishes until about 75% confluent, treated with CH223191 andPP2, alone and in combination, with DMSO as a vehicle control.

FIG. 2A shows relative light units (RLU) of C4-2 cells transfected withan XRE reporter plasmid, treated with CH223191 and PP2, alone and incombination, with DMSO as a vehicle control.

FIG. 2B shows relative light units (RLU) of C4-2 cells transfected withan ARE reporter plasmid treated with CH223191 and PP2 alone and incombination, with DMSO as a vehicle control.

FIG. 3A shows qRT-PCR analysis of AhR mRNA expression in C4-2 prostatecancer cells treated with CH223191 and PP2, alone and in combination,with DMSO as a vehicle control.

FIG. 3B shows qRT-PCR analysis of CYP1B1 mRNA expression in C4-2prostate cancer cells treated with CH223191 and PP2, alone and incombination, with DMSO as a vehicle control.

FIG. 3C shows qRT-PCR analysis of AR mRNA expression in C4-2 prostatecancer cells treated with CH223191 and PP2, alone and in combination,with DMSO as a vehicle control.

FIG. 3D shows qRT-PCR analysis of KLK3 mRNA expression in C4-2 prostatecancer cells treated with CH223191 and PP2, alone and in combination,with DMSO as a vehicle control.

FIG. 4A shows proliferation of C4-2 prostate cancer cells treated withCH223191 and PP2, alone and in combination, with DMSO as a vehiclecontrol.

FIG. 4B shows percentage of cells from C4-2 prostate cancer cellstreated with CH223191 and PP2, alone and in combination, with DMSO as avehicle control.

In the Figures, CH223191 was abbreviated to CH223 for formattingpurposes.

DETAILED DESCRIPTION Definitions

The terms “a” and “an” are defined as one or more unless this disclosureexplicitly requires otherwise. The term “substantially” is defined aslargely but not necessarily wholly what is specified (and includes whatis specified; e.g., substantially 90 degrees includes 90 degrees andsubstantially parallel includes parallel), as understood by a person ofordinary skill in the art. In any disclosed embodiment, the terms“substantially,” “approximately,” and “about” may, but need not, besubstituted with “within [a percentage] of” what is specified, where thepercentage includes, for example, any of 0.1, 1, 5, and 10 percent.

The feature or features of any embodiment described herein may beapplied to other embodiments, even though not described or illustratedexpressly together, unless expressly prohibited by this disclosure orthe nature of the embodiments.

As used throughout, by a “subject” is meant a living being. Thus, the“subject” can include domesticated animals, such as cats, dogs, etc.,livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratoryanimals (e.g., mouse, rabbit, rat, guinea pig, etc.) and birds. In someembodiments, the subject is a mammal, such as a primate, for example, ahuman.

“Amount effective” and “effective amount,” in the context of acomposition or dosage form for administration to a subject, refers to anamount of the composition or dosage form that produces one or moredesired responses in the subject, for example, preventing proliferationof prostate cancer in patients. Therefore, in some embodiments, anamount effective is any amount of a composition provided herein thatproduces one or more of these desired responses. The amount is one thata clinician believes to have a clinical benefit for a prostate cancersubject in need of cancer prevention or treatment.

Effective amount can involve only improving the patient's condition,although in some embodiments, it involves restoring patient's condition.An amount that is effective can also be an amount of a compositionprovided herein that produces a desired therapeutic endpoint or adesired therapeutic result. Administration to a subject of an effectiveamount of the compositions disclosed herein results in cancer treatmentor prevention in the subject. The achievement of any of the foregoingare monitored by routine methods.

In various embodiments of the compositions and methods provided, theeffective amount is one in which the subject is symptom-free, such ascancer-free, for at least 1 week, at least 2 weeks, at least 1 month, atleast 2 months, at least 3 months, at least 4 months, at least 5 months,at least 6 months, at least 9 months, at least 1 year, at least 2 years,at least 5 years, or longer. In other embodiments of compositions andmethods provided, the effective amount is one which produces ameasurable desired response, for example, a measurable decrease ordisappearance of cancer in the patient for at least 1 week, at least 2weeks, at least 1 month, at least 2 months, at least 3 months, at least4 months, at least 5 months, at least 6 months, at least 9 months, atleast 1 year, at least 2 years, at least 5 years, or longer.

An effective amount can depend on the particular subject being treated;the severity of a condition, disease or disorder; the individual patientparameters including age, physical condition, size and weight; theduration of the treatment; the nature of concurrent therapy (if any);the specific route of administration, other conditions or diseases thatthe subject may have, and like factors within the knowledge andexpertise of the health practitioner.

The effective amount of an aryl hydrocarbon receptor (AhR) antagonist inthe therapeutic composition can be, for example, 0.1-500 mg per kg of aliving subject, 0.1-1 mg/kg, 1-5 mg/kg, 6-10 mg/kg, 11-20 mg/kg, 21-30mg/kg, 31-40 mg/kg, 41-50 mg/kg, 51-60 mg/kg, 61-70 mg/kg, 71-80 mg/kg,81-90 mg/kg, 91-100 mg/kg, 101-110 mg/kg, 111-120 mg/kg, 121-130 mg/kg,131-140 mg/kg, 141-150 mg/kg, 151-160 mg/kg, 161-170 mg/kg, 171-180mg/kg, 181-200 mg/kg, 201-225 mg/kg, 226-250 mg/kg, 251-275 mg/kg,276-300 mg/kg, 301-325 mg/kg, 326-350 mg/kg, 351-375 mg/kg, 375-400mg/kg, 401-425 mg/kg, 426-450 mg/kg, 451-475 mg/kg, 476-500 mg/kg,0.2-400 mg/kg, 0.5-300 mg/kg, 1-250 mg/kg, 2-200 mg/kg, 5-150 mg/kg,10-100 mg/kg, 20-80 mg/kg, 30-70 mg/kg, 40-60 mg/kg, or 45-65 mg/kg.

The reduction of proliferation of the prostate cancer cells disclosedherein means that at least 50% of the prostate cancer cells in a livingsubject has been abolished, for example, 50-52.5%, 52.5-55%, 52.5-57.5%,57.5-60%, 60-62.5%, 62.5-65%, 65-67.5%, 67.5-70%, 70-72.5%, 72.5-75%,75-77.5%, 77.5-80%, 80-82.5%, 82.5-85%, 85-87.5%, 87.5-90%, 90-92.5%,92.5-95%, 95-97.5%, 97.5-99%, 99-99.5%, or 99.5-100%.

The predetermined interval to effect chronical treatment results canmean, for example, every day to every year, every two days, every threedays, every four days, every five days, every six days, every week,every 1.5 week, every two weeks, every 2.5 weeks, every three weeks,every 3.5 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7weeks, every 8 weeks, every 9 weeks, every 10 weeks, every 11 weeks,every 12 weeks, every 13 weeks, every 14 weeks, every 15 weeks, every 16weeks, every 17 weeks, every 18 weeks, every 19 weeks, every 20 weeks,every 21 weeks, every 22 weeks, every 23 weeks, every 24 weeks, every 25weeks, every 26 weeks, every 27 weeks, every 28 weeks, every 29 weeks,every 30 weeks, every 31 weeks, every 32 weeks, every 33 weeks, every 34weeks, every 35 weeks, every 36 weeks, every 37 weeks, every 38 weeks,every 39 weeks, every 40 weeks, every 41 weeks, every 42 weeks, every 43weeks, every 44 weeks, every 45 weeks, every 46 weeks, every 47 weeks,every 48 weeks, every 49 weeks, every 50 weeks, every 51 week, or every52 weeks.

“Dosage form” means a pharmacologically and/or immunologically activematerial in a medium, carrier, vehicle, or device suitable foradministration to a subject.

“Pharmaceutically acceptable excipient”, or varients thereof (e.g.,‘pharmaceutically excipient”) means a pharmacologically inactivematerial used together with the liposomes disclosed herein and carriersto formulate the compositions disclosed herein. Pharmaceuticallyacceptable excipients comprise a variety of materials known in the art,including but not limited to saccharides (such as glucose, lactose, andthe like), preservatives such as antimicrobial agents, reconstitutionaids, colorants, saline (such as phosphate buffered saline), andbuffers.

Throughout this application, various publications are referenced.Reference to the publications is not made to indicate that applicantfinds the entire contents thereof, accurate, but because at least someof the publications describe aspects of relevant state of the art.

The present disclosure may be understood more readily by reference tothe following detailed description of embodiments including furtherreference to the Figures.

General

Altered c-SRC activity has been implicated in the development, growth,progression, and metastasis of human cancers including prostate cancer.SRC is known to regulate several biological functions of tumor cells,including proliferation. There are several SRC inhibitors underevaluation for clinical effectiveness, but they have shown littleactivity in monotherapy trials of solid tumors. Combination studies arebeing explored by in vitro analysis and in clinical trials.

Simultaneous inhibition of the aryl hydrocarbon receptor (AhR) and theSRC abolishing androgen receptor (AR) signaling in prostate cancer cellsis disclosed herein as an effective treatment for prostate cancer. AhRhas been reported to interact with the SRC signaling pathway duringprostate cancer development. The c-SRC protein kinase is associated withthe AhR complex in the cytosol and upon ligand binding to AhR, c-SRC isactivated and released from the complex. AhR has also been shown toregulate AR signaling, which remains functionally important in thedevelopment and progression of prostate cancer.

Co-inhibition of AhR and SRC is shown herein to inhibit AR activity.Evaluation of total protein and cellular fractions revealed decreasedpAR expression and AR nuclear localization. Assays utilizing andandrogen responsive element (ARE) and qRT-PCR analysis of AR genesrevealed decreased AR DNA binding and transcriptional activity in thepresence of both AhR and SRC inhibitors. Furthermore, co-inhibition ofAhR and SRC reduced the growth of prostate cancer cells compared toindividual treatments. However, the present disclosure is the first todisclose simultaneous inhibition of AhR and SRC to inhibit AR signalingand prostate cancer cell growth.

The SRC-family tyrosine kinases (SFKs) are oncogenic enzymes thatcontribute to the initiation and progression of many types of cancer,including prostate cancer. SRC plays an important role in cellproliferation, differentiation, adhesion, and migration. SRC has beenidentified as a potent and specific therapeutic target for prostatecancer progression.

During progression to an androgen-independent state, prostate cancercells continue to express the androgen receptor (AR) andandrogen-regulated genes, indicating that the AR is critical for theproliferation of castration-resistant prostate cancer (CRPC) cells. CPRCis defined by rising prostate-specific antigen (PSA) levels orprogressive disease in the setting of serum testosterone levels withinthe castrate range. CRPC appears to continue to rely on the AR forgrowth and progression.

AR is a member of the steroid hormone receptor family which is primarilyresponsible for mediating the physiological effects of androgens bybinding to specific DNA sequences, known as androgen response elements(AREs). The AR protein is phosphorylated at multiple serine/threonineresidues and phosphorylation at some of these sites has been purportedto regulate nuclear localization and export. Tyrosine phosphorylation ofAR protein by non-receptor tyrosine kinases SRC may have a role in ARactivation in the low androgen environment, thereby promoting thedevelopment of CRPC. SRC-mediated phosphorylation of AR at Y534 resultedin the activation of AR and nuclear translocation and DNA binding in theabsence of androgen.

Moreover, SRC has also been reported to interact with other pathways,such as the aryl hydrocarbon receptor (AhR) signaling pathway duringprostate development. The c-SRC protein kinase is associatedspecifically with the AhR complex along with hsp90 in the cytosol and,following ligand binding to the Ah-receptor sub-unit, c-SRC is activatedand released from the complex. AhR is constitutively active in advancedprostate cancer cell lines that model CRPC and where SRC activity isalso elevated. AhR helps to sustain androgen-independent growth ofprostate cancer cells. Attenuation of AhR activity reduces expression ofphosphorylated AR, androgen responsive genes and androgen mediatedgrowth. Rapid activation of c-SRC kinase following treatment with an AhRligand has been reported in several different cell lines and may berequired for AhR mediated regulation of AR activity.

SRC is highly expressed in PCa cell lines, as well as in the majority ofPCa specimens. SRC inhibitors have recently reached the clinicaldevelopment stage in managing patients with metastatic PCa. However, SRCinhibitors have shown little activity in monotherapy trials andcombination studies are being conducted to further evaluate the effectof SRC inhibition solid tumors. The importance of c-SRC kinase activityfor aryl hydrocarbon receptor (AhR) signaling has been demonstrated andmay identify AhR as a target in combination therapy. Disclosed hereinare compositions and methods of co-targeting AhR and SRC as an effectivestrategy to abolish uncontrolled AR activity in CRPC.

Tyrosine kinase inhibitors (TKIs) have been extensively studied as atreatment for multiple malignancies. SRC is one of the TKIs regarded asa scaffolding adaptor between membranes and/or intracellular proteinsand these interactions can result in mutual activation/repressiondepending on phosphorylation exchanges. SRC activation has been observedin several cancers, including PCa. Through both direct and indirectinteraction with the AR, SRC is able to reinforce the proliferative andantiapoptotic actions of the AR, even in the absence of specificligands. These molecular mechanisms constitute a solid rationale infavor of the use of SRC inhibitors in routinely managing patients withPCa. However, monotherapy designed clinical trials using SRC inhibitorshave had limited success and combination therapy may prove morebeneficial.

Considering SRC is an integral component of the cytosolic AhR complex,AhR activity may lead to persistent phosphorylation of AR by SRC kinaseeven in the presence of SRC inhibitors. Co-immunoprecipitationexperiments revealed that AhR forms a protein complex with SRC andregulates activity by phosphorylating SRC (Tyr416) and dephosphorylatingSRC (Tyr527).

Immunoprecipitation assays revealed the association of AR with SRC,suggesting complex formation among them. Other studies have shown thatSRC kinase can cause AR transactivation in C4-2 PCa cells. Inhibition ofSRC kinase function with a specific inhibitor resulted in decreased ARactivation. There is previous evidence that SRC can facilitate crosstalkbetween AhR and other transcription factors. Studies have demonstratedSRC mediated crosstalk between AhR and epidermal growth factor receptorin colon cancer cells.

The precise molecular mechanism utilized by constitutive AhR signalingto activate AR signaling requires further investigation.

As disclosed herein, co-inhibition of SRC and AhR represses AR functionin a synergistic manner. Co-inhibition of AhR and SRC with CH223191 andPP2 respectively inhibited AR phosphorylation and nuclear localization.This inhibition resulted in decreased AR transcriptional activity asevidenced by a significant decrease in the activity of an androgenresponsive element luciferase assay and expression of androgenresponsive genes. Consequently, growth of CRPC cell line, C4-2, wassignificantly inhibited by co-targeting AhR and SRC when compared toindividual inhibition of both pathways. AR signaling is essential forthe progression of prostate cancer. Simultaneous inhibition of AhR andSRC is shown here to abolish AR signaling and decrease mortalityassociated with CRPC.

Inhibitors

CH-223191 is a potent and specific aryl hydrocarbon receptor (AhR)antagonist. The chemical name for CH-223191 is1-Methyl-N-[2-methyl-4-[2-(2-methylphenyl)diazenyl]phenyl-1H-pyrazole-5-carboxamide or 2-Methyl-2H-pyrazole-3-carboxylicacid (2-methyl-4-o-tolylazo-phenyl)-amide and its structural formula isas follows.

CH-223191 inhibited TCDD-mediated nuclear translocation and DNA bindingof AhR, and inhibited TCDD-induced luciferase activity with an IC50 of30 nM. Unlike certain other AhR antagonists which display agonistactivity at high concentrations, CH-223191 did not stimulateAhR-dependent transcription even at 100 micromolar. It is also specificfor AhR, displaying no affinity for the estrogen receptor, as some otherantagonists do.

PP2 is a selective inhibitor of SRC-family tyrosine kinaseswith >10,000-fold selectivity over ZAP-70 and JAK2. The chemical namefor PP2 is4-Amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo[3,4-d]pyrimidine or4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine and itsstructural formula is as follows.

Pharmaceutical Carriers/Delivery of Pharmaceutical Products

The aryl hydrocarbon receptor (AhR) antagonist and a SRC inhibitordescribed herein can be administered in a pharmaceutically acceptablecarrier. By “pharmaceutically acceptable” is meant a material that isnot biologically or otherwise undesirable, i.e., the material may beadministered to a subject without causing any undesirable biologicaleffects or interacting in a deleterious manner with any of the othercomponents of the pharmaceutical composition in which it is contained.The carrier would naturally be selected to minimize any degradation ofthe active ingredient and to minimize any adverse side effects in thesubject, and would be well known to one of skill in the art.

The compositions may be administered in any suitable way, for example,parenterally (e.g., intravenously), by intramuscular injection, byintraperitoneal injection, or the like.

The exact amount of the compositions required will vary from subject tosubject, depending on the species, age, weight and general condition ofthe subject, the severity of the disorder being treated, its mode ofadministration and the like. Thus, it is not possible to specify anexact amount for every composition. However, an appropriate amount canbe determined by one of ordinary skill in the art using only routineexperimentation given the teachings herein.

Parenteral administration of the composition, if used, is generallycharacterized by injection. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution of suspension in liquid prior to injection, or asemulsions. A more recently revised approach for parenteraladministration involves use of a slow release or sustained releasesystem such that a constant dosage is maintained. See, e.g., U.S. Pat.No. 3,610,795.

The aryl hydrocarbon receptor (AhR) antagonist and a SRC inhibitor maybe in solution or suspension (for example, incorporated intomicroparticles, liposomes, or cells).

Suitable carriers and their formulations are well known in the art.Typically, an appropriate amount of a pharmaceutically-acceptable saltis used in the formulation to render the formulation isotonic. Examplesof the pharmaceutically-acceptable carrier include, but are not limitedto, saline, Ringer's solution and dextrose solution. The pH of thesolution is from about 5 to about 8, for example, from about 7 to about7.5. It will be apparent to those persons skilled in the art thatcertain carriers may be more preferable depending upon, for instance,the route of administration and concentration of composition beingadministered.

Pharmaceutical carriers are known to those skilled in the art. Thesemost typically would be standard carriers for administration of drugs tohumans, including solutions such as sterile water, saline, and bufferedsolutions at physiological pH. Other compounds will be administeredaccording to standard procedures used by those skilled in the art.Pharmaceutical compositions may include carriers, thickeners, diluents,buffers, preservatives, surface active agents and the like in additionto the molecule of choice. Pharmaceutical compositions may also includeone or more active ingredients such as antimicrobial agents,anti-inflammatory agents, anesthetics, and the like. The pharmaceuticalcomposition may be administered in a number of ways depending on whetherlocal or systemic treatment is desired, and on the area to be treated.Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

EXAMPLES

Chemical and Reagents: AhR antagonist, (CH223191) was purchased fromSigma Aldrich. SRC kinase inhibitor, protein phosphatase 2 (PP2) waspurchased from Sigma Aldrich.

Cell Culture: Adherent monolayer cultures of C4-2 human prostate cancercell lines, Clark Atlanta University, Atlanta, Ga., were maintained inRPMI 1640 medium supplemented with 10% FBS. Cells were grown at 37° C.with 5% CO2 in humidified atmosphere, and media was replaced every otherday. Cells were split (1:3), when they reached near confluence.

Protein Isolation and Western Blot Analysis: Protein samples wereisolated using the Thermo Scientific NE-PER Extraction kit for cellularfractions or commercially available cell lysis buffer (Cell Signaling)for total protein. Protein samples were resolved by SDS-PAGE andtransferred to a PVDF membrane. Immunoblotting was carried out with 200μg/ml mouse AhR monoclonal antibody at 1:500 dilution in 5% milk, 200μg/ml mouse AR monoclonal antibody at 1:50 dilution in 5% milk, 100μg/ml mouse pAR monoclonal antibody at 1:50 dilution in 5% milk, and 100μg/ml rabbit pSRC monoclonal antibody at 1:1000 dilution in 5% BSA.Blots were washed three times (10 min each) with TBST. The blots werethen incubated in 1:2500 dilution of secondary antibody and washed threetimes (15 min each) with TBS. Bands were visualized with an enhancedchemiluminescence (ECL) kit as specified by the manufacturer. Multipleexposures of each set of samples were produced. The relativeconcentration of target protein was determined by computer analysisusing image J and normalized to an internal standard (topoisomerase,β-tubulin, β-actin).

RNA Extraction and Quantitative qRT-PCR Analysis: Total RNA was isolatedfrom cell monolayers grown in 100 mm tissue culture dishes using RNeasyMini Kit (Qiagen). 2 μg of the total RNA was reverse-transcribed usingthe Superscript II kit (Invitrogen), according to the manufacturer'srecommendations. The cDNA served as a template in a 25 μl reactionmixture and was processed using the following protocol: an initialdenaturation at 95° C. for 3 min, followed by 39 amplification cycles(95° C. for 10 s and 55-65° C. for 30 s), 95° C. for 10 s, 65° C. for 5s and 95° C. for 50 s. The 25 μl qPCR reaction mixture was mixed withGoTaq qPCR Master Mix (Promega). Melt curve analyses were performedafter each run to ensure a single product. Relative gene expression wasdetermined using the ΔΔCq calculation method. The primer sequences usedwere:

CYP1B1: Forward (5′-3′)  TGCCTGTCACTATTCCTCATGCCA & Reverse (5′-3′)TCTGCTGGTCAGGTCCTTGTTGAT. AhR Forward (5′-3′) TCCTTGGCTCTGAACTCAAGCTGT &Reverse (5′-3′) GCTGTGGACAATTGAAAGGCACGA. KLK3 Forward (5′-3′)ACTTCAGTGTGTGGACCTCCATGT & Reverse (5′-3′) AGCACACAGCATGAACTTGGTCAC. AR:Forward (5′-3′) GAGCTAGCCGCTCCAGTGCT & Reverse (5′-3′)CCTAACCAGGCGGGTCGTGG.

Primers to amplify the 470-bp cDNA fragment encoding L19 were used as aninternal control. L-19: Forward (5′-3′) TCCCAGGTTCAAGCGATTCTCCTT &Reverse (5′-3′) TTGAGACCAGCCTGACCAACATGA.

Proliferation Studies: Growth of cells was assayed using the PromegaCellTiter 96 Cell Proliferation Assay. Cells were resuspended to a finalconcentration of 1.0×10⁵/mL in RPMI. 50 μl of the cell suspension (5,000cells) was added to each well of the 96-well plate containing 50 μl ofmedia with corresponding treatment resulting in a total volume of 100μl. The micro plates were incubated at 37° C. for 24-72 hours in ahumidified, 5% CO2 atmosphere. Per manufacturer's instructions,following incubation, 20 μl of MTS/PMS solution was added to each welland incubated for 4 hours. Absorbances were read at 490 nm using theSynergy H1m multimode micro plate reader.

Cell viability studies: Viability of cells was assayed using the BiotiumXTT cell viability kit. Cells were re-suspended to a final concentrationof 1.0×10⁵/mL in RPMI. 100 μl of the cell suspension (10,000 cells) wasadded to each well of the 96-well plate. For each 96-well plate, 25 μlactivation reagent was mixed with 5 ml XTT solution to derive activatedXTT solution. Then 50 μl of this activated XTT was added to each well.The micro plates were incubated at 37° C. for 4 hours in a humidified,5% CO2 atmosphere. Absorbances were read at 490 nm and 670 nm using theSynergy H1m multimode micro plate reader. Background absorbance wassubtracted from signal absorbance to obtain normalized absorbance value.

XRE and ARE Binding: 4×10⁴ Cells were plated in a 96 well plate. C4-2cells were transfected with XRE and ARE reporter, as well as withpositive and negative control reporter plasmids using attractene. After16 hours of transfection, media was changed to standard assay media(DMEM+0.5% FBS+0.1 mM NEAA). Cells were grown for an additional 8 hoursunder normal cell conditions. After 24 hours of transfection, treat thecells and harvested cells 18 hours after treatment. A dual luciferaseassay was performed after 42 hours of transfection, and promoteractivity values are expressed as arbitrary florescence units (AFU).Experiments were performed in triplicate and the standard error isindicated.

Statistical Analysis: Each experiment was carried in triplicate and allthe values are expressed as mean+SEM. The differences between the groupswere compared by t-test or ANOVA using Instant software (GraphPadSoftware Inc., San Diego, Calif.). A value of P<0.05 was consideredstatistically significant.

Example 1 Co-Inhibition of AhR and SRC Abolishes Phosphorylation of AR

C4-2 prostate cancer cells were used as a CRPC cell model. These cellswere isolated from a chimeric tumor induced by inoculating a castratedmouse with parental androgen sensitive LNCaP cells. AhR was previouslyreported to be constitutively active in C4-2 cells and activation of SRCkinase has been verified to accompany AhR activity. C4-2 prostate cancercells were treated with AhR inhibitor (CH223191) and SRC kinaseinhibitor (PP2) alone and in combination. DMSO served as a vehiclecontrol. 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4d]pyrimidine (PP2), a widely used compound to block theactivity of SRC family kinases, reduced phosphorylation of SRC and AR inC4-2 cells. Total cellular proteins were isolated and proteins wereseparated by SDS polyacrylamine gel electrophoresis and blotted usinganti-AhR antibody, anti-AR antibody, anti-pAR antibody, anti-cSRCantibody, anti-pSRC antibody. Anti-β-actin was used as a loadingcontrol. FIG. 1 is representative of 3 independent membranes. Theaddition of specific AhR antagonist CH223191 abolished both SRC and ARphosphorylation in the presence of PP2. The expression levels ofnon-phosphorylated AR and SSRC were not affected with treatment as shownin FIG. 1A.

Nuclear AR is AhR/SRC dependent: The translocation of AR into thenucleus is solely dependent upon the phosphorylation of AR. In order toaddress the role of AhR and SRC in this process, a sub-cellularlocalization was confirmed by immunoblotting following cellularfractionation. Nuclear and cytoplasmic fractionation: C4-2 cells grownon 100 mm dishes until ˜75% confluent were treated with DMSO, CH223 andPP2 as described above. Cells were washed with cold PBS and cellularfractions were isolated per manufactures instructions using a NE-PERExtraction kit. The nuclear and cytoplasmic fractions were analyzed bywestern blotting for AhR and AR protein expression. The relative levelof cytoplasmic AhR & AR were normalized with β-tubulin expression andthe relative level of nuclear AhR & AR were normalized withtopoisomerase expression. Blots are representative of three independentexperiments. Individually, PP2 and CH223191 reduced the presence of ARin the nucleus with a greater effect shown with PP2. However,simultaneous inhibition of AhR and SRC abolished AR nuclear localizationis shown in FIG. 1B.

Example 2 Co-Inhibition of AhR and SRC Decreases Promoter Activity ofAhR and AR

The Cignal XRE and ARE luciferase reporter assays were utilized todetermine the activity of AhR and AR signaling pathways in androgenindependent (C4-2) prostate cancer cells in the presence and absence ofinhibitors. C4-2 cells were transfected with an XRE reporter plasmid &ARE reporter plasmid, as well as with positive and negative controlreporter plasmids using attractene. Following transfection, treatmentswere added to each appropriate plate. A dual luciferase assay wasperformed and promoter activity values are expressed as arbitraryflorescence units (AFU). Each bar represents mean±SEM (n=3) and wereanalyzed by student t-test. (*) denotes statistically significantdifferences (*P<0.0001). The assay showed that C4-2 prostate cancercells have a high level of AhR binding to XRE and ofAR binding to ARE inthe absence of inhibitor treatment. CH223191 reduced AhR promoteractivity by 30% while PP2 resulted in a 50% decrease in XRE bindingcompared to DMSO. Both CH223191 and PP2 reduced AR promoter activity by70% compared to the DMSO in ARE binding assay as shown in FIG. 2A andFIG. 2B respectively. Simultaneous inhibition of AhR and SRC in C4-2cells with CH223191/PP2 resulted in minimal ARE binding.

Example 3 Synergistic Inhibition of AhR and AR Target Gene Expression

To confirm the synergistic effect of CH223191 and PP2 on AhR and ARactivity, qRT-PCR was used to quantify mRNA expression of AhR responsivegenes (AhR and CYP1B1) and AR responsive genes (AR and KLK3) as shown inFIGS. 3A-3D respectively. qRT-PCR analysis examined AhR, CYP1B1, CYP1A1,AR, KLK2 and KLK3 mRNA expression in C4-2 prostate cancer cells. Cellswere treated with 50 μM of AhR inhibitor (CH223191) or 30 μM of SRCinhibitor (PP2) alone or in combination or with vehicle control (DMSO)for 72 h and total RNAs were isolated and quantitative RT-PCR wasperformed to determine the mRNA expression of each target in treatedcells. mRNA levels were normalized using L-19 which serves as aninternal control. Each bar represents mean±SEM (n=3) and were analyzedby student t-test. (*) denotes statistically significant differences(*P<0.05) compared to control.

Since AhR is constitutively active within the C4-2 cell line, CH223191reduced AhR and CYP1B1 gene expression more than twofold when comparedto the control. While PP2 alone also decreased AhR and CYP1B1 geneexpression, inhibition of gene targets was further enhanced when thecells were treated with both CH223191 and PP2. CYP1B1 gene expressionwas found to be very similar to the expression of AhR when treated witheither CH223191 or PP2 as well when both drugs were used in combination.Elevated AhR and CYP1B1 gene expression reported before tumor formationin a rat model of mammary tumorigenesis suggested differential CYP1B1regulation by a constitutively active AhR. CYP1B1 expression wasdiminished by repression of AhR activity. Because of the fundamentalrole of androgens in prostate development as well as prostate cancer,the objective was to determine if the effect of CH223191/PP2 alsoaffected gene expression of AR and downstream target gene KLK3 (PSA). 50μM of CH223191 reduced AR gene expression by 60% in C4-2 cells. Anidentical decrease in AR expression was observed in response totreatment with PP2. These results helped to confirm that these drugs notonly decrease the expression of AhR but AR as well. Additionally, whenC4-2 cells are co-treated with CH223191/PP2, AR expression is reduced by85%. Furthermore, KLK3, which encodes for the glycoprotein prostatespecific antigen (PSA), was observed to have a 65% and 70% decrease inexpression in the presence of CH223191 and PP2, respectively. Yet, whenthese two drugs were used in combination, the level of KLK3 mRNAexpression was furthered decreased to more than 97%. Therefore, it canbe concluded that these drugs have an amplified inhibitory effect on AhRand AR when used in combination.

Example 4 Simultaneous Reduction of AhR and SRC Signaling SignificantlyReduces Proliferation

Expression and activity data confirmed the effectiveness of combinationtherapy on AR. The effects were then examined on the growth of C4-2cells. The influence of AhR and SRC on androgen independent cell linegrowth was observed when C4-2 cells were grown for 24-72 hours in thepresence and absence of CH223191 and PP2. CH223191 and PP2 havesynergistic effect on proliferation on C4-2 prostate cancer cells. Cellswere grown in a 96 well plate at 5.0×10 cells per well. The cells weretreated with DMSO or 50 μM of CH223191 or 30 μM of PP2 alone or incombination for 24-72 hrs. Cell growth was measured using PromegaCellTiter 96 Cell Proliferation Assay per manufacturer instructions.Each bar represents mean±SEM (n=3), *p<0.05. B.) The cells were platedat a density of 1×10 cells/dish and exposed to DMSO or 50 μM of CH223191or 30 μM of PP2 alone or in combination for 72 hrs. Cells were harvestedand analysed for cell cycle. The cells with DMSO exposure served ascontrol. Bar graphs represent mean±SD of three separate experiments.There was no significant difference between the four treatments on therate of growth after 24 hours exposure. The growth of C4-2 cells wassignificantly inhibited in the presence of CH223191 and PP2 at 48 and 72hours. Furthermore, CH223191/PP2 demonstrated a synergistic effect ongrowth inhibition at 48-72 hours. After 72 hours of exposure to thecombination, C4-2 cells exhibited a 50% decrease in overall growth ratecompared to DMSO, as compared to a 25% decrease when CH223191 or PP2were used alone. Together these finding demonstrate co-inhibition of AhRand SRC synergistically reduce the growth rate of C4-2 PCa cells asshown in FIG. 4A. Cell cycle analysis revealed an increase in thepercent of cells in the GO/G1 phase of the cell cycle when cells weretreated with either CH223191 or PP2. The percentage of cells remainingin G0/G1 was further increased when cells were co-treated with CH223191and PP2 corresponding with the reduced growth rate seen withsimultaneous inhibition of AhR and SRC as shown in FIG. 4B.

The embodiments above are intended to be illustrative and not limiting.Embodiments are within the claims. In addition, although the presentdisclosure has been described with reference to particular embodiments,those skilled in the art will recognize that changes can be made in formand detail without departing from the spirit and scope of thedisclosure. While, as provided, publications are referenced herein toindicate aspects of the state of the art, no subject matter isincorporated that is contrary to the explicit disclosure herein.

1-14. (canceled)
 15. A therapeutic composition, for treating prostatecancer in a living subject, comprising 2-Methyl-2H-pyrazole-3-carboxylicacid (2-methyl-4-o-tolylazo-phenyl)-amide (CH223191) andAmino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), anda pharmaceutical excipient. 16-20. (canceled)
 21. The therapeuticcomposition of claim 15, wherein the 2-Methyl-2H-pyrazole-3-carboxylicacid (2-methyl-4-o-tolylazo-phenyl)-amide (CH223191) is present in anamount of about 50 μM.
 22. The therapeutic composition of claim 15,wherein Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine(PP2) is present in an amount of about 30 μM.
 23. The therapeuticcomposition of claim 15, wherein the 2-Methyl-2H-pyrazole-3-carboxylicacid (2-methyl-4-o-tolylazo-phenyl)-amide (CH223191) and theAmino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) arepresent in synergistic amounts.
 24. The therapeutic composition of claim23, wherein the 2-Methyl-2H-pyrazole-3-carboxylic acid(2-methyl-4-o-tolylazo-phenyl)-amide (CH223191) is present in an amountof about 50 μM and theAmino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) ispresent in an amount of about 30 μM.